Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

Emily Z Guo,Zhaohui Xu

doi:10.1074/jbc.m114.607903

Abstract

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.

Highlights

IST1 recruits microtubule interacting and trafficking (MIT) domain-containing proteins VPS4, LIP5, and Spartin to the mid-body during cytokinesis
IST1312–335 Is Sufficient to Bind the MIT Domains of VPS4B, LIP5, and Spartin—Mutational and NMR spectroscopic studies have previously suggested that IST1 contains two potential MIT-interacting motif (MIM) sequence motifs at its C terminus [24]
Residues 312–335 resemble a peptide sequence that could bind in the MIM1 mode, and residues 290 –308 resemble a peptide sequence that could bind in the MIM2 mode

Summary

Background

IST1 recruits MIT domain-containing proteins VPS4, LIP5, and Spartin to the mid-body during cytokinesis. Given the importance of IST1 in recruiting VPS4 and Spartin to the mid-body of dividing cells during cytokinesis, we sought to understand the structural basis of the interaction between IST1 and these different MIT domain-containing proteins. To this end, we have determined the high resolution crystal structures of the IST1 C-terminal MIM in complex with the MIT domains of VPS4, LIP5, and Spartin, which showed that IST1 binds to VPS4 and LIP5 in the MIM1 mode but to Spartin in the MIM3 mode. Using a preliminary binding code derived from this analysis, we were able to convert CHMP2A, a protein that normally binds the MIT domain only in the MIM1 mode, into one that can bind to the MIT domain of Spartin in the MIM3 mode

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION